Project Summary Zika virus (ZIKV) is a positive sense strand RNA virus of the mosquito-borne flaviviruses that recently caused a global epidemic. ZIKV infection can result in the induction of several neurological disorders, including Guillain- Barr syndrome and fetal microcephaly. Recently, a highly conserved noncoding RNA element in the 3' un- translated region of the ZIKV RNA genome called the Xrn1-resistant structure (xrRNA1) was identified that stalls the host exonuclease (Xrn1), allowing the virus to produce subgenomic flaviviral RNAs (sfRNA) directly connected to viral pathology. The crystal structure of xrRNA1 reveals that it adopts an unusual three- dimensional structure composed of a three-way junction stabilized by a pseudoknot, with the 5?-end segment threading through a helical `ring' formed by the three-way junction. This unusual structure has been shown to confound the helicase activity of Xrn1 and promote sfRNA formation. Mutations that disrupt key structural fea- tures formed by highly conserved nucleotides severely decrease Xrn1 resistance and sfRNA formation during infection. xrRNA1 is an ideal drug target in the ZIKV RNA due to the unlikelihood of generating resistance mu- tations and the presence of a druggable pocket with unique structural features that has the potential to selectively bind small molecules. This project will screen xrRNA1 against ~1 million small molecules to identify inhibitors of ZIKV replication that specifically target and disrupt the structure of xrRNA1 and thereby inhibit pro- duction of sfRNA, impacting pathogenesis by decreasing virulence. This will be accomplished using a new hybrid in silico/in vitro RNA-small molecule screening technology (RNA targeting using Experimentally-Based Ensembles for Ligand discovery? or REBEL) that makes it possible to widen RNA screens to millions of mole- cules and to create sub-libraries that are enriched with bioactive and selective RNA-targeting compounds. Aim 1 will use REBEL to identify compounds that bind and modulate the structure of xrRNA1. The one thousand top hits will be subjected to experimental screening for xrRNA1 binding and inhibition of sfRNA formation using NMR spectroscopy and in vitro sfRNA formation assays. In Aim 2, hits from in vitro assays will be tested for anti-ZIKV and anti-DENV activity and toxicity in cellular based assays. If successful, this work will identify promising leads for the development of antiviral therapies for treatment of ZIKV and may lay the foundation for a new strategy to target pathogenic flaviviruses in general.